Epithelial Transport

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					MCT09 Epithelial Transport                                              Scribe: Kathy Nguyen
November 3, 2010                                                     Reviewer: Tyler Snedden
Dr. Simon Lewis                                                  Chief Reviewer: Michael Yip
                                    Epithelial Transport

Structure of epithelium
    Six-pack Analogy
        Cans                            Epithelial cells
        Flip-top                        Apical membrane
        Bottom and sides                Basolateral membrane
        Plastic rings                   Tight junctions
        Sides of cans                   Lateral intercellular space
        Hand on which cans rest         Basement membrane
        Various types of cans in pack   Heterogenous cell types in epithelium

      3-dimensional drawing of rat proximal tubule cell demonstrates differences from six-pack
           o Convoluted borders
           o Apical membrane has brush border microvilli (↑ surface area ~20x for fluid
           o Basolateral membrane has long, columnar interdigitations
           o Mitochondria reside near basolateral membrane: near Na+/K+ ATPase, which
              needs ATP
                   Na+/K+ ATPase involved in active transport of ions from lumen to blood
                      (absorption), or blood to lumen (secretion)
      Variant nomenclature of membranes/parts based on tissue, e.g.:
           o Intestine
                   Apical- brush border or mucosa
                   Basolateral- serosa
                   Tight junction- paracellular
                            “Paracellular pathway” is composed of two series elements: (1)
                              tight junction, and (2) lateral intercellular space
           o Kidney
                   Apical- luminal
                   Basolateral- peritubular
           o Per Dr. Lewis, do not memorize the above terms now, but be aware of that they
              exist for future discussion of organ-specific discussion in the future.
      Junction types:
           o Hemidesmosomes- attach basal aspect of cell to basement membrane; provide
              mechanical support
           o Desmosomes- cell-to-cell spot welds that hold adjacent epithelial cells together
           o Gap junctions- allow intercellular electrical and chemical communication
           o Zona adherins- actin belt that lies underneath tight junctions
           o Tight junctions (formerly “zona occludens”)
                   Intermeshing web of filaments that hold cells together
                   Resistance of tight junction determined by molecular make-up (types/ratio
                      of protein strands), not the number of filaments in the junction
                   Separates two compartments
MCT09 Epithelial Transport                                              Scribe: Kathy Nguyen
November 3, 2010                                                     Reviewer: Tyler Snedden
Dr. Simon Lewis                                                  Chief Reviewer: Michael Yip
                  Occludins- tight junction component protein; interestingly, tight junction
                    function is not affected in occludin knockout mice
                  Claudins
                        Protein of tight junction which is actually responsible for acting as
                           barrier to substance movement
                        24 types
                        Ratio of claudin types determines junction resistance

Are all epithelia created equal? Do they all have equal barrier properties?
    No.
    This can be quantified experimentally using Ohms’ Law:
           o V = IR (V= voltage, I= current, R= resistance)
           o Transepithelial Resistance ranges from 5 - 70,000 Ω/cm2
                    14,000 fold difference in resistance
           o The higher the resistance, the greater ability of the epithelia to restrict
                    e.g. Proximal tubules have low resistance; urinary bladder has high
           o Spontaneous Transepithelial Voltage ranges from 0 mV to -120 mV
                    Suggests that there is active ion transport in epithelial tissue
    Hypothesized causes of resistance variability:
           o α hypothesis: High and constant junctional resistance with variable cell resistance
           o Ω hypothesis: High and constant cell resistance and variable junctional resistance
           o Sprinkler head analogy:
                    Student challenged to locate “holes” in head while blindfolded
                           Require data recording system, hose attached to a tap, and a
                              bathing suit
                           Tap attached to hose/tap, then student used finger to scan over
                              surface of sprinkler head to feel metal vs. water flow
           o Actual gallbladder experiment
                    Current passed through gallbladder epithelial tissue
                    Voltage sensed with probe at surface of tissue
                    Voltage was greater over tight junctions than cell membrane  low
                      resistance of gallbladder epithelium due to low tight junction resistance
    Epithelia divided into 2 categories:
MCT09 Epithelial Transport                                              Scribe: Kathy Nguyen
November 3, 2010                                                     Reviewer: Tyler Snedden
Dr. Simon Lewis                                                  Chief Reviewer: Michael Yip
          o Leaky epithelia
                  R= 5-100 ohms/ cm2
                  Doesn’t tend to generate spontaneous voltage
                  Water permeable because they contain high levels of aquaporins in apical
                    and basolateral membranes
                  Can only maintain low concentration gradient, because ions leak
                  Good transporters of electrolytes and water
          o Tight epithelia
                  R= 500-70000 ohms/cm2
                  More likely generate a large voltage
                  Tends to be water impermeable due to lower number of aquaporins
                  Can maintain a large concentration gradient (e.g. stomach epithelium
                    separates pH 1-2 (lumen) and pH ~7 (blood)- 106 difference)
     Common characteristics of all epithelia
          o Na+/K+ ATPase in the basolateral membrane
                  Except retinal pigment epithelium and
                    choroids plexus epithelium, where it is in
                    the apical membrane instead
                  Pumps 2 K+ into cell and 3 Na+ out;
                    requires 1 ATP  interior relatively
                    electrically negative
          o Potassium channel in basolateral membrane (with
             same exceptions as above)
          o Chloride is lower inside of cell
          o Cell pH slightly more acidic than interstitial space
          o Voltage of -50 to -70 mV (primarily dependent upon K+ gradient)
          o All apical membranes are composed of lipids, but protein content differs, as they
             determine the transport properties of the epithelia

Sodium Transport
    Ussings’ frog skin experiment:
         o Observed sheet of viable skin from ventral surface of frog suspended between two
            compartments (“blood” and “pond”) of NaCl bath:
                 Resistance: 5000 Ω/cm2;
                 Transepithelial potential (Vt): -20 to -120 mV (blood side 0 mV)
         o Active transport of what ion generates this Vt?
                 Replaced Cl- with gluconate  no change in voltage
                         Conclusion: Cl- transport not responsible for Vt
                 Replaced Na+ with choline  voltage goes to 0 mV
                         Suggests Na+ transport (from blood to pond) responsible for Vt
                         Measured, and active ion flow across epithelium was equal to net
                           isotopic Na+ flux
                 Added ouabain (which inhibits Na+/K+ ATPase) to blood side  voltage
                    slowly decreased to zero
                         Inhibition of Na+/K+ ATPase causes loss of membrane potential
         o Conclusion: Na+ transport is responsible for ion gradient and membrane potential
MCT09 Epithelial Transport                                                 Scribe: Kathy Nguyen
November 3, 2010                                                        Reviewer: Tyler Snedden
Dr. Simon Lewis                                                     Chief Reviewer: Michael Yip
     Sodium Absorption Mechanism (modified version of the Ussings’ observations)
          o Na+ enters cell (down its                        Sodium Absorption
             concentration gradient)           Lumen                                         Blood
             through conductance channels                 K+
             in apical membrane
          o Once in cell, 3 Na+ are                  Na+            Na+

             extruded across basolateral
             membrane by Na+/K+ ATPase,                                       ATP
             in exchange for 2 K+                                                       K+
               +                                                             K+
          o K passively diffuses down its
             concentration gradient) via K+
             channels in the basolateral
             membrane  cell interior
             electrically negative
                  Chemical and electrical gradients both favor Na+ influx
          o Net movement of Na+  blood side electrically positive, and lumen electrically
                  Favors Cl- movement from lumen to blood via paracellular tight junctions
                  Small amount of K+ leaks back out via tight junctions
          o Net Na+ and Cl- absorption, and positively charge blood compartment
          o Model found in distal and collecting duct of kidney, lung (neonatal), distal colon,
             salivary ducts, sweat ducts, taste receptors, and urinary bladder

Properties of epithelial sodium channels (ENaC)
    Composed of 3 subunits (α, β, and γ)
    Each subunit has two transmembrane domains
    Inhibited by amiloride (diuretic, which “acts like a plug in the opening of a bottle”)
    Channel activity and density in membrane increased by aldosterone
    Pathophysiology
          o Gain of function (increased number of functional channels)
                   Either by extended duration of channel opening or ↑ density of channels
                           Especially in distal tubule and collecting duct
                   Effects- hypertension, hypernatremia, hypokalemia, metabolic alkalosis
                           Aldosterone stimulates Na+ absorption and K+ secretion, especially
                              in distal tubules of kidney and colon
                           K+ shift promotes cells to take up protons from blood
                   Causes:
                           Hyperaldosteronism
                                  o Primary aldosteronism (Conn’s syndrome)- hyperplasia or
                                      adrenal adenocarcinoma
                                  o Secondary aldosteronism, caused by ↑ renin secretion
                           Liddle’s syndrome- trafficking problem of sodium
                                  o α and β subunits have mutation inhibits channel
                                      internalization, ubiquitination, and degradation
                                             ↑ channel density
MCT09 Epithelial Transport                                             Scribe: Kathy Nguyen
November 3, 2010                                                     Reviewer: Tyler Snedden
Dr. Simon Lewis                                                 Chief Reviewer: Michael Yip
          o Loss of function (decreased number of function channels)
                  Effects- hypotension, hyponatremia, and hyperkalemia
                  Causes:
                        Pseudohypoaldosteronism type 1
                               o Aldosterone content normal, but tissue fails to react,
                                       Tissue lack aldosterone receptors
                                       Na+ channels mutated to that they do not conduct
                        Addison’s disease
                               o Atrophy of adrenal cortex  ↓ aldosterone secretion
                               o John F. Kennedy had Addison’s disease
          o Long term aldosterone action increases the density of sodium channels in apical
             membranes and potassium pumps in basolateral membrane

Isosmotic fluid absorption
     Diamond’s observations on gallbladder
           o Initial observations
                   Transepithelial resistance: 50 Ω/cm2
                   Transepithelial potential (Vt): 0 mV
           o These values do not fit Ussings’ model- is the tissue dead?
                   The function of the gallbladder is to concentrate bile by transporting NaCl
                      and water from lumen to blood (absorption)
                          Therefore if the tissue is still alive, weight should change over time
           o Filled gallbladder with fluid and in a beaker of NaCl solution then measured
              weight change in gallbladder sac over time (using gravimetric method)
                   Results:
                          Weight decreased over time
                          No change in composition or osmolality inside sac
                          Suggests active ion and water absorption
           o Weight loss of sac over time with inhibited by removing Na+ or Cl- from bathing
           o Ouabain in blood side compartment inhibited weight loss
           o Concluded Na+/K+ ATPase must be involved in isosmotic fluid absorption

                                                 Sodium Chloride Absorption
Model for NaCl absorption- in kidney and intestine
   Na / H Exchanger:
         +  +

          o Na+ enters cell
                                          Lumen                          Blood
          o Proton leaves
   HCO3-/ Cl- Exchanger:                     Na       Na  +                 +

          o Cl- enters cell           H CO 2
                                                       H   +         +
                                                                             Na                              +
                                                           H                             +

          o Bicarbonate ion exits            HCO      HCO   +  3
                                                                    ADP      3
   Proton and bicarbonate ion form H O        2
                                               Cl     Cl       -

                                        +                                     K                                  +
      carbonic acid in lumen           CO          2  CO  H CO   Cl      2           2           3

                                                                    H2O c.a.
MCT09 Epithelial Transport                                                 Scribe: Kathy Nguyen
November 3, 2010                                                        Reviewer: Tyler Snedden
Dr. Simon Lewis                                                    Chief Reviewer: Michael Yip
     Carbonic acid dissociates into CO2 and water
     CO2 diffuses into cell
     CO2 combines with water to form carbonic acid inside cell
     Carbonic acid dissociates into bicarbonate ion and proton
     Products are recycled, giving continuous movement of Na+ and Cl+ into cell
     Cl- exits cell via chloride channel in basolateral membrane (down its concentration
     Na+/Cl- cotransporter (not shown) moves Na+ and Cl- out of cell
     Na+ out of and K+ in via Na+/K+ ATPase in basolateral membrane
     K+ then diffuses out of the cell through K+ channels in basolateral membrane
     Net sodium and chloride absorption without net charge movement
          o This is the second reason leaky epithelia do not generate voltage

Water transport
    General concept: water follows osmolality gradients, even small differences in osmolality
       can greatly influence water permeability
    e.g. When glucose absorbed from lumen along with Na+ (secondary active transport since
       glucose is going down its concentration gradient), intracellular osmolality increases
           o Causes water to enter cell from lumen by osmosis
           o Tight junctions restrict diffusional space between cells
           o Na+/K+ ATPase and facilitative diffusion  Na+ and glucose into interstitial space
              and are trapped
           o Water flows to interstitial space by osmosis                 ...
                                                                        ….. H2O Water Transport
           o Increased flow of water and intracellular solutes
              cause initial interstitial space expansion
           o Further increase in transport and build up of
              hydrostatic pressure  flushing of ions and                    ….
              water down length of lateral space through
              basement membrane and into capillaries
    Requires miniscule osmotic gradients (2 mOsm across
                                                                             H2 O
       apical membrane, 1 across basolateral  total of only
           o Affects membranes highly permeable to water
              because of aquaporins
    Occurs in ileum, proximal segments of colon, proximal
       tubules of kidney, salivary duct cells, and gallbladder
Ion secretion
    Small and large intestine can absorb and secrete fluids
    Composition of secreted fluid is an isosmotic NaCl solution
    Secretion is controlled by neurotransmitters, hormones, and bacterial toxins (e.g. cholera)
    Stimulation of secretion in the intestine causes an increase in the transepithelial voltage
       (lumen goes more negative)
    Secretion is inhibited by ouabain and removal of blood side Na+, K+, and Cl-
    Model of Intestinal Cl- secretion:
           o Cl- enters cell through a 2Cl-/ Na+/ K+ cotransporter
MCT09 Epithelial Transport                                                 Scribe: Kathy Nguyen
November 3, 2010                                                        Reviewer: Tyler Snedden
Dr. Simon Lewis                                                     Chloride Secretion
                                                         Intestinal Chief Reviewer: Michael Yip
          o Na+/ K+ ATPase removes 3
             Na+ and brings in 2 K+             Lumen      K   +                            Blood
               +                  +
          o K leaves through K channel
          o Makes cell interior negative               -
          o Cl- leaves via Cl- channel in                                              2 Cl    -
                                                                             Cl  -
                                                                                       Na  +
             apical membrane (down
                                                                                        Na     +
             electrochemical gradient, but                                   ATP
             against its concentration                                      K+

                  Cystic fibrosis                     Na  +

                     conductance regulator (CFTR)- common Cl- channel
                           Regulated by phosphorylation by protein kinase A
          o Makes lumen negative, attracting sodium
          o Na+ enters lumen through tight junctions following electrochemical gradient
                  Small amount of K+ also attracted
          o Increased osmotic pressure  water flow  ↑ hydrostatic pressure  bulk flow
             of near isosmotic fluid into lumen
     CFTR found in:
          o Pancreas
          o Sweat ducts
          o Parotid gland
          o Submaxillary glands
          o Small salivary glands
          o Intestinal glands
          o Intrahepatic bile duct
          o Gallbladder
          o Tracheobronchial glands
          o Brunners glands
     CFTR Properties
          o 2 transmembrane regions, each with 6 transmembrane segments and joined by a
             nucleotide binding domain
                  Phosphorylation by PKA activates the channel
          o Chloride channel
     Pathophysiology
          o Diarrhea diseases
                  Cholera causes irreversible activation of CFTR
                           Can cause as much as ~24 L diarrhea/day
                  Cholera toxin has 4 subunits
                           Alpha subunit enters cells and cause ribosilation of adenylate
                             cyclase  increases cAMP
                           Ribosilation is irreversible so channels are always phosphorylated
                           Causes a massive secretion of NaCl into lumen and subsequent
                           Toxin also inhibits cellular proton exchanger
          o Cystic fibrosis
MCT09 Epithelial Transport                                             Scribe: Kathy Nguyen
November 3, 2010                                                    Reviewer: Tyler Snedden
Dr. Simon Lewis                                                 Chief Reviewer: Michael Yip
                  Cl- channel mutation (over 200 possible mutations cause various levels of
                  Over 70% caused by mutation of alanine at position 508 in cystic fibrosis
                  Inhibits trafficking of channel to apical membrane
                  Affects
                         Pancreas- becomes fibrotic because enzymes can’t enter
                           duodenum; enzymes digests pancreas inside
                         Trachea- mucous becomes very viscous because it can’t secrete
                           NaCl and water  mucus can’t move as easily  bacterial
                           infections occur more easily

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